Question

Differential Display

Polymerase Chain Reaction

Template DNA

SpecificityMust be complementary to flanking sequences of target regionNot be complementary to other non-target regions of genomeNested polymerase chain reaction (nested PCR): to reduce non-specific binding Primer LengthToo short  lack specificityToo lon

Product SizeBasic Taq polymerase can easily amplify fragments up to 1,000 to 2,000 bpTypical qPCR amplicons are 70-200 bp in lengthToo long  Fluorescent intensity not in linear rangePrimer DimersIf the primers have self-complementary sequences, the prime

General Considerations in Primer Design

General Considerations in Primer Design

G/C ContentPrimers should be about 50% G/C (40~60%) Not have long runs of G/C or A/T (>3 bp) A stretch of A/T’s might only weakly base pairA stretch of G/C might promote mis-annealingG/C clampTo ensure the stability of this interaction, primers are often

Web Based Tools for Primer Design

http://biotools.nubic.northwestern.edu/OligoCalc.html

Web Based Tools for Primer Design

https://www.ncbi.nlm.nih.gov/tools/primer-blast/

Question

Rapid Amplification of cDNA Ends (RACE)

Rapid Amplification of cDNA Ends (RACE)

Multiplex PCR

Primer Design Assistant

PCR-based Techniques: Subtractive hybridization

QuestionCan RNA be amplified directly by PCR? Why and why not?

Reverse Transcription PCR

Real Time - PCR

Real Time - PCR

Amplification Plot